This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Successful dissemination of HIV-1 in infected individuals depends on efficient transmission of viral particles from infected (producer) to uninfected (target) cells. In vitro propagation studies have established that HIV-1 particles are most effectively transmitted to target cells if they bud at the so-called virological synapse (VS) that forms between producer and target cells. Such synaptic virus transmission is most likely also prevalent in vivo, when HIV-1 spreads in secondary lymphoid organs of infected individuals. Hitherto, the events leading to the formation, maintenance and disassembly of the VS are poorly understood. Previously, we and other laboratories have established that HIV-1 particles exit from cells at membrane segments that are enriched in tetraspanins. These cellular integral membrane proteins are known to function as organizers of plasma membrane-based processes, including cell-cell fusion and adhesion, but their roles in virus replication, particularly during the late stages, remain to be elucidated. While some of our preliminary data suggest that the presence of tetraspanins at the virological presynapse may be beneficial for the virus, other available evidence suggests that these host cell proteins restrict virus spread. We thus hypothesize that HIV-1 evolved to spatially and temporally regulate tetraspanin expression levels such that it can disseminate efficiently. Altogether, these studies will provide further insight into the molecular mechanisms underlying HIV-1 spread and thus pathogenesis, and they may contribute knowledge that can be used for the development of anti-viral strategies. In the Specific Aims of this proposal we propose: 1) To analyze how tetraspanins inhibit HIV-1-induced membrane fusion, thus allowing cell-to-cell transfer without fusion of producer and target cell. 2) To investigate how, overall, tetraspanins in HIV-1 producer cells affect the transmission of viral particles to target cells. 3) To examine the kinetics, determinants and potential consequences of HIV-1-induced tetraspanin downregulation in infected cells.